The clinical usefulness of any chemotherapeutic agent or drug can be severely affected by the emergence of cellular resistance to that drug. Accordingly, a significant amount of research has been conducted in an attempt to elucidate the cellular mechanisms involved with resistance to drugs, as well as methods for overcoming such resistance. To date a number of putative cellular mechanisms involved in drug resistance have been proposed. These include:
(i) altered metabolism of the drugs, which could include decreased activation or increased deactivation;
(ii) impermeability of the target cell or organism to the active compound;
(iii) altered specificity of an inhibited enzyme;
(iv) increased production of a target molecule;
(v) increased repair of cytotoxic lesions; and
(vi) bypassing of an inhibited reaction by alternative biochemical pathways.
The cellular mechanisms involved in drug resistance are complex, and consequently there has been little progress in the development of generally applicable methods for overcoming drug resistance problems. A further complicating factor is that while drug resistance is a problem associated with nearly all chemotherapeutic applications, it is more often associated with diseases which require on-going and prolonged treatment with a number of concurrent drugs.
In cancer treatment, for example, impermeability of the cancer cells to the active compound is often observed. Moreover, it is often found that resistance to one drug may confer resistance to other biochemically distinct drugs. This has been termed multidrug resistance. Drugs that are typically affected by the multidrug resistance problem include doxorubicin, vincristine, vinblastine, colchicine and actinomycin D. In at least some cases, multidrug resistance is a complex phenotype that has been linked to a high level of expression of a cell membrane drug efflux transporter called MdrI protein, also known as P-glycoprotein. This membrane “pump” has broad specificity, and acts to remove from the cell a wide variety of chemically unrelated toxins (see Endicott et al., 1989).
Recently, a similar mechanism of broad-spectrum drug resistance has been reported for certain microorganisms. These results indicate the existence of bacterial efflux systems of extremely broad substrate specificity that are similar to the multidrug resistance pump of mammalian cells (see Nikaido, 1993).
Substances which reverse multidrug resistance are known as resistance modification agents (RMAs), and are of importance in potentiating the cytotoxicity of chemotherapeutic agents to which a human cancer has become resistant. Although many agents have been identified as RMAs in vitro, a large proportion of these have little or no therapeutic potential because of high toxicity in vivo at the doses required to reverse multidrug resistance. For example, metabolic poisons, such as azide, are able to reverse multidrug resistance in vitro, but have no usefulness in vivo. Most other highly effective RMAs, such as PSC833, appear to work as competitive antagonists of a drug-binding site on the MdrI protein. Many of these agents also have toxicity that limits their usefulness in vivo. Consequently, there is a need to develop alternate pharmacological strategies for reversing multidrug resistance.
In an attempt to overcome poor tumour uptake of anti-neoplastic drugs and reduce systemic toxicity (Singh et al., 1991), investigators have attempted to target anti-neoplastic drugs such as methotrexate (MTX) to the location of malignant tissue. Several studies have already attempted to target chemotherapeutic agents to tumours by linking drugs to polymers selected for their affinity to tumours (Hudecz et al., 1993; Klein et al., 1994; Akima et al., 1996). However, while these polymers may help to deliver active agents to their target tissues, they do not necessarily overcome the drug resistance problem.
One polymer which has been proposed as a tumour targeting agent is hyaluronan (HA). HA, also known as hyaluronic acid, is a naturally occurring polysaccharide comprising linear-chain polymers, which is found ubiquitously throughout the animal kingdom. HA is highly water-soluble, making it an ideal drug delivery vehicle for biological systems.
The applicants have now surprisingly found that HA exhibits unique structural and physicochemical properties which not only enhance its usefulness as a drug carrier, but also aid in overcoming drug resistance. In high concentrations of ˜1 g/L, HA adopts a stiffened random coil configuration that occupies an exceptional volume relative to molecule mass (Laurent, 1970), and at this level or below, it forms loose links to macromolecular networks (FIG. 1). While not wishing to be bound by any particular theory, we consider that, based on the physical characteristics of HA, the mechanism of interaction between polysaccharide and agents such as methotrexate, may take one or both of two forms:
(i) Chemical Interaction (FIG. 2A).
Ionic bonding could occur between the MTX amine groups and the HA carboxyl groups, but such an interaction could cause precipitation. Another possible interaction is via hydrogen bonding between available amine groups on the drug and hydroxyl groups of the HA, but this is unlikely because methotrexate is relatively insoluble in water; therefore if this were to occur it would be a very weak interaction. The most likely bonding between MTX and HA would be via hydrophobic interactions between MTX's numerous hydrophobic groups and the hydrophobic patches in the secondary structure of HA (Scott et al., 1989).
(ii) Molecular Association.
Where MTX is merely “mixed” in HA gel (FIG. 2B) with no specific chemical bond formation, MTX could become entrapped within the 3-dimensional meshwork formed by higher concentrations of HA (Mikelsaar and Scott, 1994), so that the drug simply diffuses from the HA after administration. If HA is rapidly taken up and bound by specific cell receptors, the drug will be released in higher concentration at these points eg. lymph nodes, liver, bone marrow, tumour cells with HA receptors.
While again not wishing to be bound by any particular theory, one mechanism by which HA helps to target active agents may be via the characteristic over-expression of HA receptors in several tumour types (Stamenkovic et al., 1991; Wang et al., 1998). The HA receptors CD44, Receptor for Hyaluronan Mediated Motility (RHAMM) and ICAM-1, have been linked to tumour genesis (Bartolazzi et al., 1994) and progression (Günthert 1993; Arch et al., 1992). RHAMM is a major factor in mediating tumour cell motility and invasion (Hardwick et al., 1992). It has been demonstrated that RHAMM is required for H-ras transformation of fibroblasts (Hall et al., 1995), which would make this receptor a potential participant in tumour formation and growth. ICAM-1, a receptor tentatively linked to HA metabolism (McCourt et al., 1994), is highly expressed in transformed tissues such as mouse mastocytomas (Gustafson et al., 1995a) and in the stroma and clusters of tumour cells of human breast carcinomas (Ogawa et al., 1998).
Increased expression of HA receptors on tumour cells provides a rationale for attempting the incorporation of HA into chemotherapeutic treatment regimens. However, the very limited data obtained to date actually teach away from the presently claimed process. For example, limited success has been obtained by chemically complexing HA to mitomycin C and epirubicin; investigators were able to inhibit colon carcinoma growth by 0.8-25% (Akima et al., 1996). Klein and colleagues (1994) reported an increased uptake of the drug into implanted rat mammary and Fischer bladder carcinomas was achieved by merely mixing HA with 5-FU. Mouse mastocytomas were also demonstrated to have an affinity for intravenously-injected HA (Gustafson et al., 1995b).
However, while some of the HA research has shown that HA can be used as a drug carrier, none of this research has shown that HA is capable of overcoming cellular resistance to drugs.